CN105039239A - Cell transformation induction liquid and use thereof - Google Patents
Cell transformation induction liquid and use thereof Download PDFInfo
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- CN105039239A CN105039239A CN201510397799.7A CN201510397799A CN105039239A CN 105039239 A CN105039239 A CN 105039239A CN 201510397799 A CN201510397799 A CN 201510397799A CN 105039239 A CN105039239 A CN 105039239A
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Abstract
The invention relates to a cell transformation induction liquid and a use thereof. The cell transformation induction liquid comprises niacinamide and an epidermal growth factor. Niacinamide can promote cell propagation conversion and the epidermal growth factor can promote UC-MSC propagation and islet-like cell cluster formation. Therefore, the cell transformation induction liquid can induce production of a lot of islet cells transformed from UC-MSC, and the produced islet cells have high quality and can secrete much insulin. The cell transformation induction liquid has an important meaning for UC-MSC clinical transplant for diabetes mellitus type 1 treatment.
Description
Technical field
The present invention relates to biotechnology, organizational project and biomedicine field, the cell transformation induced liquid particularly for by UC-MSC Induction Transformation being islet cells.
Background technology
Diabetes be insulin secretion relatively or the defect such as definitely not enough or the insulin receptor sugar, fat and the protein metabolism disorder disease that cause.Clinically, I type and part ii patients with type Ⅰ DM many employings subcutaneous insulin injections are treated.And cell therapy is the ideal treatment strategy of diabetes especially insulin-dependent diabetes mellitus (IDDM), especially for the diabetic subject losing islet cell function, implanting the islet cells of excreting insulin or its surrogate to be optimal methods for the treatment of.At present, islet cell transplantation treatment diabetes achieve some curative effects, but donor's cells originates, the difficulty such as deficiency, serious immunological rejection significantly limit the application of this therapy.
Stem cell has extremely strong self-renewal capacity and multi-lineage potential, is the desirable target cell of gene therapy.Stem cell is divided into myeloid-lymphoid stem cell (as embryonic stem cell, the all histocytes of body can be divided into), multipotential stem cell born of the same parents (have multi-lineage potential, Various Tissues cell can be divided into, as mescenchymal stem cell etc.) and specially can stem cell (maintaining the single direction self of a certain particular organization cell, as gut epithelial stem cells etc.).The characteristic that the continuous breakthrough of stem cells technology and stem cell itself have makes the mankind likely cultivate some stem cell in vitro, directional induction its be divided into various histocytes required for us, or be used for organizational project for needed for clinical as seed cell, Stem Cell Engineering with this end in view relates to most of difficult medical problem of human body nearly all vital tissue organ and facing mankind, as the treatment of cardiovascular disorder, diabetes, malignant tumour, bone and cartilage defect, senile dementia, Parkinson's disease, burn, Spinal injury and hereditary defect etc.Due to the multipotential stem cell in people's amniotic fluid, Cord blood, peripheral blood and marrow, have the advantages such as multiplication capacity is strong, abundance, collection convenience, this type of stem cell transplantation has played important effect in the treatment of disease in the blood system, malignant tumour, autoimmune disorder etc.
Suitable genetic modification is carried out to stem cell, make it the cell becoming energy excreting insulin, it is one of ideal strategy for the treatment of insulin-dependent diabetes mellitus (IDDM), and be in islet cells process by stem cell transformation, induced liquid plays vital effect, for the induction mode that stem cell is different, required induced liquid is different.But, the method that induced dry-cell the most frequently used in prior art is converted into islet cells is specially to insulin-like cell conversion for applying the insert Tissue Culture Dish external evoked UC-MSC of Dual culture plate (i.e. umbilical cord mesenchymal stem cells): choose the 3rd generation UC-MSC, with the L-DMEM substratum re-suspended cell containing 10%FBS (foetal calf serum), adjustment cell density is 1 × 105/ml, be inoculated in common 6 orifice plates, under inverted microscope observation of cell adherent after, change L-DMEM/RPMI1640 (1:1) substratum (substratum that low-sugar type improvement Du Shi Eagle's medium and RPMI16401:1 mix) into, to be separated the islet cells of acquisition simultaneously, be inoculated in Dual culture plate with 50/hole, carry out the Dual culture of UC-MSC and islet cells two kinds of cells.But, this kind of co-culture method not only needs certain islet cells source, process is more loaded down with trivial details, spend larger, and mostly more being of obtaining is dispersed in insulin-like cell, comparatively small amt, islet cells group does not almost have, and the insulin-like cell be dispersed in is relatively little, amount of insulin secretion is difficult to reach clinical transplantation requirement less.At present, investigator is also had to study a kind of method of UC-MSC directly being induced nesidioblast, therefore, in the urgent need to a kind of for UC-MSC directly being induced the induced liquid of nesidioblast.
Summary of the invention
Technical problem to be solved by this invention is, the islet cells obtained for induced liquid induction UC-MSC and islet cells Dual culture mode in prior art is less, comparatively small amt, and spend the defects such as larger, a kind of induced liquid that UC-MSC can be converted into islet cells is provided.
The technical solution adopted for the present invention to solve the technical problems is: a kind of cell transformation induced liquid, described induced liquid comprises nicotinamide and Urogastron.
In cell transformation induced liquid provided by the invention, in described induced liquid, the concentration of nicotinamide is the concentration of 5-15mmol/L and Urogastron is 15-30 μ g/L.
In cell transformation induced liquid provided by the invention, described induced liquid also comprises β-nerve growth factor and the 1-10nmol/L activin A of 60 μ g/L-150 μ g/L.
In cell transformation induced liquid provided by the invention, described induced liquid also comprises reductive agent.
In cell transformation induced liquid provided by the invention, described reductive agent is β-coloured glaze base ethanol, and β-coloured glaze base ethanol is 5-15 μ g/L.
In cell transformation induced liquid provided by the invention, in described induced liquid, the concentration of nicotinamide is 5mmol/L, and the concentration of Urogastron is 30 μ g/L, and the concentration of β-nerve growth factor is 150 μ g/L, the concentration of activin A is the concentration of 10nmol/L, β-coloured glaze base ethanol is 10 μ g/L.
In cell transformation induced liquid provided by the invention, in described induced liquid, the concentration of nicotinamide is 15mmol/L, and the concentration of Urogastron is 15 μ g/L, and the concentration of β-nerve growth factor is 60 μ g/L, the concentration of activin A is the concentration of 1nmol/L, β-coloured glaze base ethanol is 15 μ g/L.
In cell transformation induced liquid provided by the invention, in described induced liquid, the concentration of nicotinamide is 10mmol/L, and the concentration of Urogastron is 25 μ g/L, and the concentration of β-nerve growth factor is 100 μ g/L, the concentration of activin A is the concentration of 4nmol/L, β-coloured glaze base ethanol is 5 μ g/L.
The present invention protects above-mentioned cell transformation induced liquid to be converted into application in islet cells process at UC-MSC further.
Implement cell transformation induced liquid provided by the invention and application thereof, following beneficial effect can be reached: this induced liquid can while maintenance UC-MSC cytoactive, UC-MSC is promoted to increase and propagation conversion, promote that later stage UC-MSC changes into islet cells group and increases islet cells, the method existing UC-MSC of improvement and islet cells Dual culture being obtained to islet cells has great importance; To be converted into the application in islet cells process at UC-MSC from induced liquid provided by the invention, induced liquid provided by the invention not only saves the process that existing induction method mid-early stage obtains islet cells, and the islet cell mass making UC-MSC change into is more, and islet cells quality is better, can secrete more Regular Insulin.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 cell distribution maps that to be the islet cells that adopts cell transformation induced liquid provided by the invention induction UC-MSC to change into dyeed under the microscope that obtains by dithizone;
Fig. 2 cell distribution enlarged view that to be the islet cells that adopts cell transformation induced liquid provided by the invention induction UC-MSC to change into dyeed under the microscope that obtains by dithizone.
Embodiment
Less for solving the islet cells that in prior art, induced liquid induction UC-MSC and islet cells co-cultivation mode obtain, and the defects such as comparatively small amt, innovative point of the present invention is to provide a kind of cell transformation induced liquid, can promote that UC-MSC changes into islet cells, this induced liquid can promote cell proliferation, increase, thus achieves the object improving UC-MSC transformation efficiency and improve the rear islet cells quality of conversion.
Particularly, cell transformation induced liquid for UC-MSC being converted into islet cells provided by the invention comprises: nicotinamide and Urogastron, preferably, in induced liquid, the concentration of nicotinamide is the concentration of 5-15mmol/L and Urogastron is 15-30 μ g/L.The Main Function of nicotinamide participates in respiratory chain, participates in respiratory and the electron transfer process of cell proliferation conversion, promotes that the propagation of cell transforms; Urogastron can promote UC-MSC to breed and promote the formation of Islet-like clusters; Therefore, can promote that the propagation of UC-MSC transforms by induced liquid provided by the invention, prepare for UC-MSC is converted into islet cells, promote the formation of islet cells group.
Further, induced liquid provided by the invention also comprises β-nerve growth factor and the 1-10nmol/L activin A of 60 μ g/L-150 μ g/L.β-nerve growth factor belongs to neurotrophic factor, can promote the growth of UC-MSC, maintains the normal active and function of UC-MSC; And activin A, the propagation of UC-MSC can be promoted, therefore, in induced liquid, add the activity that β-nerve growth factor and activin A not only can keep UC-MSC, improve survival rate, but also the propagation of UC-MSC can be promoted further.
In addition, owing to transforming period in cell proliferation, the oxygenizement of cell is stronger, oxyradical can be discharged in substratum, when oxyradical runs up to a certain degree, cytolemma and organoid film can be destroyed, kill cell, be unfavorable for the growing multiplication of cell, cell is made not reach very high density, therefore, reductive agent can also be comprised in induced liquid provided by the invention, as β-coloured glaze base ethanol, beta-mercaptoethanol is a kind of strong reductant, the oxyradical accumulated in cell culture medium can be neutralized, cell fission can be made like this to arrive very high density and not dead, thus be conducive to the propagation of UC-MSC, the oxygenizement that can reduce cell period is transformed in cell proliferation, improve the level of glutathion inside cell, scavenging activated oxygen, be conducive to the conversion of UC-MSC, preferably, the concentration of beta-mercaptoethanol is 8-10 μ g/L.
Cell transformation induced liquid provided by the invention, being applied to the method steps that UC-MSC is converted into islet cells is:
Get third generation UC-MSC, be placed in incubator, and according to the following steps third generation UC-MSC is induced:
1) in cell culture medium, add the first induced liquid, be cultured to cell and complete breeding, obtain pre-inversion stem cell body;
2) in the DMEM/F12 substratum of Insulin-Transferrin-selenium containing massfraction being 1%-5%, add the second induced liquid, cultivate above-mentioned pre-inversion stem cell body and terminate to cell transformation, obtain islet cells liquid;
Wherein, the first induced liquid is the induced liquid for UC-MSC being converted into islet cells provided by the invention, under induced liquid provided by the invention promotes, UC-MSC is constantly bred, promote the formation of cell mass, for forming a large amount of islet cellss and islet cells group prepares;
The second induced liquid comprises 5-15mmol/L nicotinamide and 5-15 μ g/L Prostatropin.
Nicotinamide participates in respiratory and the electron transfer process of cell proliferation conversion, promotes that the propagation of cell transforms; Prostatropin can promote accelerated cellular proliferation, and cell volume increases; DMEM/F12 substratum containing Insulin-Transferrin-selenium can impel the stem cell body orientation of conversion to change into islet cells.
Compared with prior art, adopt induced liquid provided by the invention, can transform UC-MSC proceed step by step, make UC-MSC directly can change into islet cells, and transform rear islet cells without the need to UC-MSC and islet cells Dual culture being obtained, and adopt induced liquid provided by the invention can promote propagation and the increase of UC-MSC cell, promote the formation of islet cells group, improve the transformation efficiency of islet cells.
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
Application the invention provides the method that UC-MSC is converted into islet cells by cell transformation induced liquid and comprises the following steps:
(1) separation and Culture of UC-MSC
The umbilical cord of the healthy fetus of full-term pregnancy Cesarean esction is got under aseptic condition, every milliliter is soaked in containing after carrying out 4-6h process in the physiological saline of 25U heparin sodium after umbilical cord is in vitro, the arteriovenous of umbilical cord is rejected in super clean bench, peel off outer amnion, umbilical cord be cut into the tissue block of 1mm × 1mm × 1mm size and be affixed at the bottom of culture dish, the low sugar DMEM that to add containing volume fraction be 10% foetal calf serum is placed in 37 DEG C, 5%CO2 saturated humidity incubator cultivates, remove after cultivating 2 weeks, reach when 80%-90% converges until attached cell and carry out Secondary Culture.
Wherein, low sugar DMEM (dulbecco'smodifiedeaglemedium, the improvement Du Shi Eagle's medium) composition being 10% foetal calf serum containing volume fraction is: containing 10% foetal calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, IL-3,5ug/L granular leukocyte colony stimulating organism factor of 15ug/L, 1.0-3.3g/L glucose.
It is appreciated of course that the source mode of third generation UC-MSC is also not limited thereto, in prior art, retrievable third generation UC-MSC is all applicable to the present invention.
(2) Induction Transformation of UC-MSC
1) in volume fraction be 30% foetal calf serum DMEM/F12 substratum in add containing 150 μ g/L β-nerve growth factors, 10nmol/L activin A, 5mmol/L nicotinamide, 30 μ g/L Urogastrons, the first induced liquid of 10 μ g/L β-coloured glaze base ethanol, in 37 DEG C of incubators with 5%CO2, cultivate 7d complete breeding to cell, obtain pre-inversion stem cell body;
2) add containing 10mmol/L nicotinamide, the second induced liquid of 10 μ g/L Prostatropins, with the DMEM/F12 substratum containing massfraction being 1% Insulin-Transferrin-selenium, in 37 DEG C of incubators with 5%CO2, cultivate 14d to pre-inversion stem cell body terminate to cell transformation, obtain islet cells liquid;
(3) separation of islet cells
Discard above-mentioned nutrient solution supernatant, 2 times are cleaned with phosphate solution (calling PBS damping fluid in the following text), add L-DMEM (low-sugar type improvement Du Shi Eagle's medium, glucose concn is 1.0-3.3g/L) cultivate 1-2h after, collect supernatant, PBS washes 2 times, be changed to H-DMEM (high glycoform improvement Du Shi Eagle's medium again, glucose concn is 3.15-4.5g/L) cultivate 1-2h, collect supernatant, be the islet cells after separation.
Embodiment 2
This embodiment compared with embodiment 1, the Induction Transformation step 1 except third generation UC-MSC) to some extent difference except, other process steps are all identical.
In this embodiment, the Induction Transformation step 1 of third generation UC-MSC) be:
Add containing 60 μ g/L β-nerve growth factors, 1nmol/L activin A, 15mmol/L nicotinamide, 15 μ g/L Urogastrons, the first induced liquid of 15 μ g/L β-coloured glaze base ethanol, volume fraction is cultivate in the DMEM/F12 substratum of 5% foetal calf serum to complete breeding to cell in 7 days, obtains pre-inversion stem cell body.
Embodiment 3
This embodiment compared with embodiment 1, the Induction Transformation step 1 except UC-MSC) to some extent difference except, other process steps are all identical.
In this embodiment, the Induction Transformation step 1 of UC-MSC) be:
Add containing 100 μ g/L β-nerve growth factors, 4nmol/L activin A, 10mmol/L nicotinamide, 25 μ g/L Urogastrons, the first induced liquid of 5 μ g/L β-coloured glaze base ethanol, be cultivate in the DMEM/F12 substratum of 10% foetal calf serum to complete breeding to cell in 7 days in volume fraction, obtain pre-inversion stem cell body.
For verifying that induced liquid provided by the invention is converted into the beneficial effect played in islet cells process at UC-MSC further, to in embodiment of the present invention 1-3 obtain the mensuration that islet cells carries out relevant parameter respectively, mainly comprise the mensuration of islet cells rate, C peptide secretory volume and amount of insulin secretion; And by arranging the outstanding beneficial effect of the present invention of control group contrast, control group is the islet cells obtained by utilizing induced liquid of the prior art to induce UC-MSC and islet cells, refers to specification sheets background technology part of the present invention.
Concrete mensuration process is:
1, Flow cytometry islet cells percentage is adopted
Cell culture supernatant after inducing 17d to the islet cells liquid of inducing in embodiment of the present invention 1-3 and control group respectively proceeds as follows respectively: after being 0.25% tryptic digestion with massfraction, PBS buffer solution 3 times, the centrifugal 5min of 1000rpm, abandon supernatant, add insulin-CY5 (insulin C Y5 fluorescent mark Su Su), Flow cytometry 105 cells; Carry out 6 groups of parallel laboratory test islet cellss respectively to be marked by insulin-CY5, the software analysis carried by flow cytometer can draw the data of islet cells rate in table 1.
2, radioimmunology is utilized to detect Regular Insulin and the C-peptide content of islet cells secrete
Cell culture supernatant after inducing 17d to the islet cells liquid of inducing in embodiment of the present invention 1-3 and control group respectively proceeds as follows respectively: draw culture supernatant 0.5ml in transfer pipet ,-20 DEG C of frozen specimen takens.According to the insulin human of Beijing Fu Rui bio-engineering corporation and C peptide radioimmunological kit, the content measuring Regular Insulin and C peptide in specimen taken is described, measurement result is as shown in table 1.
Table 1:
As shown in Table 1, by adopting the islet cells rate that the invention provides cell transformation induced liquid induction UC-MSC conversion much larger than prior art, and from C peptide secretory volume and amount of insulin secretion, islet cells utilization ratio after conversion is higher, therefore, induced liquid provided by the invention not only increases the quantity of islet cells, also improves the quality of islet cells.
In addition, by arrange dithizone staining be verified further adopt cell transformation induced liquid provided by the invention can improve islet cell mass and obtain islet cells group; This tested object is: 1, experimental group-embodiment of the present invention 1 obtains islet cells; The islet cells of 2, control group-induce with UC-MSC in prior art and islet cells Dual culture mode acquisition, refers to specification sheets background technology part of the present invention;
Concrete steps are:
1, the two sulphur track working fluid of preparation: 50mg dithizone is dissolved in 5 milliliters of DMSO (dimethyl sulfoxide (DMSO)), filtration, packing are stored in-20 DEG C of preservations.
2, experimental group and control group are washed 2 times with phosphate solution respectively, respectively add the phosphate solution of l milliliter and the dithizone working fluid (final concentration 1%, V/V) of 10 μ L, hatch 15min for 37 DEG C, islet cells coloring case is observed, respectively as depicted in figs. 1 and 2 under inverted microscope.In Fig. 1, dark parts represents the islet cells induced by method provided by the invention, and as can be seen from the figure, islet cells is assembled agglomerating, and area is comparatively large, and quantity is more; Fig. 2 dark parts represents the islet cells enlarged view induced by prior art, as can be seen from the figure islet cells comparatively disperse, not agglomerating, islet cell mass is relatively less.
In sum, cell transformation induced liquid provided by the invention, make UC-MSC while maintenance cytoactive, can promote that UC-MSC breeds to transform, promote that UC-MSC increases simultaneously, prepare for UC-MSC changes into islet cells group and increases islet cells, the method existing UC-MSC of improvement and islet cells Dual culture being obtained to islet cells has great importance; To be converted into the application in islet cells process at UC-MSC from induced liquid provided by the invention, induced liquid provided by the invention not only saves the process that existing induction method mid-early stage obtains islet cells, and it is more easy to operate to make UC-MSC be converted into the process of islet cells, cost can be reduced simultaneously, and final to obtain islet cell mass more, and islet cells quality is better, can secrete more Regular Insulin, significant for clinical transplantation UC-MSC treatment type 1 diabetes.
Below by reference to the accompanying drawings embodiments of the invention are described; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protect, also can make a lot of form, these all belong within protection of the present invention.
Claims (9)
1. a cell transformation induced liquid, is characterized in that: described induced liquid comprises nicotinamide and Urogastron.
2. cell transformation induced liquid according to claim 1, is characterized in that, in described induced liquid, the concentration of nicotinamide is the concentration of 5-15mmol/L and Urogastron is 15-30 μ g/L.
3. cell transformation induced liquid according to claim 2, is characterized in that, described induced liquid also comprises β-nerve growth factor and the 1-10nmol/L activin A of 60 μ g/L-150 μ g/L.
4. cell transformation induced liquid according to claim 3, is characterized in that, described induced liquid also comprises reductive agent.
5. cell transformation induced liquid according to claim 4, is characterized in that, described reductive agent is β-coloured glaze base ethanol, and β-coloured glaze base ethanol is 5-15 μ g/L.
6. cell transformation induced liquid according to claim 5, it is characterized in that, in described induced liquid, the concentration of nicotinamide is 5mmol/L, the concentration of Urogastron is 30 μ g/L, the concentration of β-nerve growth factor is 150 μ g/L, the concentration of activin A is the concentration of 10nmol/L, β-coloured glaze base ethanol is 10 μ g/L.
7. cell transformation induced liquid according to claim 5, it is characterized in that, in described induced liquid, the concentration of nicotinamide is 15mmol/L, the concentration of Urogastron is 15 μ g/L, the concentration of β-nerve growth factor is 60 μ g/L, the concentration of activin A is the concentration of 1nmol/L, β-coloured glaze base ethanol is 15 μ g/L.
8. cell transformation induced liquid according to claim 5, it is characterized in that, in described induced liquid, the concentration of nicotinamide is 10mmol/L, the concentration of Urogastron is 25 μ g/L, the concentration of β-nerve growth factor is 100 μ g/L, the concentration of activin A is the concentration of 4nmol/L, β-coloured glaze base ethanol is 5 μ g/L.
9. the cell transformation induced liquid according to any one of claim 1-8 is converted into the application in islet cells process at UC-MSC.
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